Center for Veterinary Biologics SAM 119.04

Testing Protocol Page 1 of 8

Supplemental Assay Method for Titration of Pseudorabies Virus Neutralizing Antibody (Constant Virus- Varying Serum Method)

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 119

Supplemental Assay Method for Titration of Pseudorabies Virus Neutralizing 

Antibody (Constant Virus-Varying Serum Method)

Date: August 7, 2023

Number: SAM 119.04

Supersedes: SAM 119.03, April 19, 2017

Standard Requirement: 9 CFR part, 113.133

Contact: Email: Methodsrequest.notification@usda.gov

Phone: Center for Veterinary Biologics, 515-337-6100

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
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Supplemental Assay Method for Titration of Pseudorabies Virus Neutralizing Antibody 

(Constant Virus-Varying Serum Method)

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Table of Contents

1. Introduction

2. Materials

2.1 Cell cultures

2.2 Growth medium

2.3 Indicator virus

2.4 Diluent

3. Preparation for the Test

3.1 Preparation of reagents/control procedures

3.2 Preparation of the sample

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions

Appendices
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Supplemental Assay Method for Titration of Pseudorabies Virus Neutralizing Antibody 

(Constant Virus-Varying Serum Method)

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro assay method which uses plaque 

reduction in a cell culture system to determine the antibody titer of serum against Pseudorabies 

virus.

2. Materials

2.1 Cell cultures

Multiple 6-well disposable plastic plates (35-mm well diameter, Linbro Multi-Dish 

Disposo-Trays) are seeded with Madin-Darby Bovine Kidney (MDBK) cells free of 

extraneous agents, at a cell count that will produce a monolayer after 2 days of 

incubation, e.g., 3 mL/well of cells split 1 to 4 in 10% fetal bovine serum (FBS)-

supplemented growth medium.

2.2 Growth medium

The cells are grown in Minimum Essential Medium (MEM) with additives (Appendix I) 

at a temperature of 35°- 37°C in an incubator containing an atmosphere of 5% carbon 

dioxide (CO2) and a relative humidity of 70% to 80%. Growth medium is not changed 

unless acidity occurs or cells are not growing well.

2.3 Indicator virus

National Veterinary Services Laboratories (NVSL) reference PRV virus is used as the 

indicator virus.

2.4 Diluent

Diluent (Appendix II), without serum, is used to make dilutions.

3. Preparation for the Test

3.1 Preparation of reagents/control procedures

3.1.1 Dilution of test serum

The serum is heat-treated at 56°C for 30 minutes. Serial twofold dilutions are 

made in sterile tubes containing diluent and mixed using a Vortex or similar type 
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Supplemental Assay Method for Titration of Pseudorabies Virus Neutralizing Antibody 

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mixer. For example, twofold dilutions can be made as follows:

1. 0.5 mL diluent is added to tubes 2, 3, 4, and 5.

2. 0.5 mL serum is added to tubes 1 and 2. The pipette is discarded and 

tube 2 is mixed. Tube 1 contains 0.5 mL of the undiluted serum, and tube 

2 contains a 1:2 dilution of serum.

3. 0.5 mL from tube 2 is transferred to tube 3. The pipette is discarded 

and tube 3 is mixed. Serum in tube 3 is a 1:4 dilution.

4. This process is continued until the desired number of serum dilutions 

are made. 0.5 mL is discarded from the last dilution tube.

3.1.2 Dilution of indicator virus

A vial of NVSL PRV reference virus is thawed, mixed, and diluted to contain 30 

to 70 plaque forming units (PFU) per 0.1 mL. This dilution is determined by 

previous titrations and is designated the “stock virus.”

3.2 Preparation of the sample

Serum neutralization of virus and virus control.

3.2.1 An equal volume of stock virus (0.5 mL) is added to each serum dilution 

tube, mixed, and incubated at 37°C for 1 hour to allow for neutralization of virus. 

This mixing of equal volumes of serum dilution and virus results in a further 

twofold dilution of serum. Thus, the undiluted serum (tube 1) becomes a 1:2 final 

dilution, the initial 1:2 becomes 1:4, etc.

3.2.2 Virus controls are prepared by making serial tenfold dilutions of stock 

virus in diluent. Dilutions of 100

, 10-1, and 10-2 are incubated at 37°C for 1 hour, 

in the same way as the virus-serum mixture.

4. Performance of the Test

4.1 Inoculation of cells and virus adsorption

Before inoculation of the MDBK cell monolayers, the growth medium is removed by 

aspiration with a sterile Pasteur pipette attached to a vacuum tube, or by decantation.
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Supplemental Assay Method for Titration of Pseudorabies Virus Neutralizing Antibody 

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Two wells are inoculated with 0.2 mL of each virus-serum mixture. Four wells are 

inoculated with 0.1 mL of the virus-diluent mixture. 

In each series of tests, 2 or more wells containing cell monolayers are maintained as 

uninoculated controls.

Inoculated cells are placed in a 3-5% CO2 incubator at 37°C for 1 hour to allow for virus 

adsorption. During the incubation, plates are gently rocked and rotated every 20 minutes 

to redistribute inoculum.

4.2 Overlay and incubation

Following incubation, the plates are ready for the overlay, which serves as maintenance 

media for the cells. Three mL of the overlay medium (Appendix III) at room 

temperature are added to each well and the plates are returned to the CO2 incubator. The 

plates remain in the incubator undisturbed for 4 days.

4.3 Plaque counting

The cultures are prepared and counted as follows.

4.3.1 Overlay medium is poured off.

4.3.2 One or 2 mL crystal violet solution are added to each well and allowed to 

spread evenly over the monolayers. The plates are allowed to stand at room 

temperature for a minimum of 10 minutes.

4.3.3 The crystal violet solution is washed from the cell monolayers by dipping 

each plate several times in a container of running cold tap water. The plates are 

dried.

4.3.4 Plaques are counted and recorded. The plaques are visible as circular areas 

in the monolayer where cells have been destroyed by the virus and fail to retain 

the dye as the uninfected cells do.

5. Interpretation of the Test Results

The plaque reduction titer is the highest serum dilution which causes a 50% reduction in the 

virus plaque count as compared to the average plaque count of the virus-diluent mixture. 

Example: Stock virus plaque count is 50. The serum-virus dilution with half the plaques (24) is 

the titer of the serum.
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Supplemental Assay Method for Titration of Pseudorabies Virus Neutralizing Antibody 

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6. Report of Test Results

Record all test results on the test record.

7. References

Title 9, Code of Federal Regulations, part 113.333, U.S. Government Printing Office, 

Washington, DC. 

8. Summary of Revisions 

Version .04 

• Updated coversheet, contact list and minor changes within document. 

Version .03

• The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.

Version .02 

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document: 

• The Contact information has been updated. 
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Supplemental Assay Method for Titration of Pseudorabies Virus Neutralizing Antibody 

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Appendices

Appendix I

Growth Medium

Lactalbumin hydrolysate or Edamin 0.5%

MEM (Eagle) with Earles salts q.s. ad 100.00%

Sodium bicarbonate 2.2 g

Gentamicin sulfate 50 mcg per mL

Penicillin 25 U per mL

Streptomycin 100 mcg per mL

Heat-inactiviated or irradiated fetal bovine serum 10.0%

L-Glutamine 1.0%

Appendix II

Diluent

Lactalbumin hydrolysate or Edamin 0.5%

MEM (Eagle) with Earles salts q.s. ad 100.0%

Sodium bicarbonate 2.2 g

Gentamicin sulfate 50 mcg per mL

Amphotericin B 5.0 mcg per mL

Penicillin 100 U per mL

Streptomycin 100 mg per mL

L-Glutamine 1.0%
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Supplemental Assay Method for Titration of Pseudorabies Virus Neutralizing Antibody 

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Appendix III

Overlay (gum tragacanth)

Preparation of 2% gum tragacanth:

Tragacanth is added (2.0 g/100 mL for 2%), a little at a time, to the desired 

volume of deionized water and mixed vigorously with a blender.

This is sterilized by autoclaving at 15 lbs/psi for 15 minutes. It is then stored at 

4°C.

The viscosity of tragacanth does not seem to vary significantly with temperature 

change. The 2% tragacanth can be made up in large volumes and stored at 4°C 

until used.

Use of 2% gum tragacanth:

The needed volume of 2% tragacanth is added to an equal volume of 2X medium. 

This mixture is warmed to room temperature before adding to cell cultures.